Method of preparing leucine



amino acids resulting from Patented July 30, 1935 UNITED STATESmrrnonorrnsranmo LEUCINE Harold M. Barnett,

signor to S. a corporation of Ohio M. A. Corporation,

Garfield Heights, one, a.

Cleveland, Ohio,

no Drawing. Application May 1, 1933, Serial No. 668,809

This invention relates to a method of obtaining leucine and, moreparticularly, to a new and improved method of obtaining this amino acidfrom mixtures with other acids in a pure or substantially purecondition.

Leucine, CsHraNOz, or alpha amino isocaproic acid, is one of the aminoacids obtainable by hydrolysis of proteins, in considerable amountdepending upon the protein selected as a source. Since the amino acids,as a group, are generally considered to be among the most important,both biologically and chemically, in the realm of organic compounds,leucine is seen to belong to a class of substances of great interest.However, the known methods for the preparation of leucine, practicedonly in the laboratory, are long and tedious and often give-a productwhich is badly contaminated with other amino acids. Consequently, theprice has been very high, at present $12.00 for 10 grams, or theequivalent of about $540.00 per pound, with the result that experimentalinvestigations as to its properties, derivatives, physiological eifects,etc., are the chief uses to which it has been put. A less expensive andmore emcientmethod of preparation which wouldplace leucine on'th'emarket in large quantities in sufliciently pure condition is, naturally,a prerequisite to the development of practical uses for the substance.

It is, accordingly, an objector the present invention to provide amethod for the preparation of leucine eihciently in pure orapproximately pure condition. Another object of the invention is toprovide an improved method for the separation of leucine from mixturescontaining other the hydrolysis of proteins. Other objects of theinvention will in part be obvious and in part appear hereinafter.

v The process is applicable either to naturally occurring or syntheticmixtures of amino acids, such as the protein hydrolysates, or othermaterials containing leucine. Amino acids are obtained from proteinsupon the breaking down of the latter by hydrolysis with acids, alka ifisor enzymes. Acids are more commonly employed, the usual method being tohydrolyze with an acid, such as hydrochloric acid, neutralize and thencollect a mixture of amino acids in one form or another. Thispreliminary hydrolysis is, of course, omitted when the leucine is to beseparated from existing amino acid mixtures.

In the new process, where protein isselected as the starting material,protein materials, such as wheat; gluten, casein, zein andthe like arehydrolyzed by boiling the some with hydrochloric 1 clai s. (or. 260-119)acid, specific gravity about 1.1, for about sixteenhours, the ratio ofacid to protein being three to one. It should be understood, however,that any and all oi. the conditions during this hydrolysis step such as,for example, time, tem- 5 perature, strength of acid, use of catalysts,ratio of-acid to protein, etc., may be varied to suit any conditionswhich may arise. For instance, hydrolysis under pressure may be used toreduce the time of treatment, and the product of hydrol 10 ysis by acidsother than hydrochloric, alkalies or enzymes may be used in the process.In fact, any feasible method may be used to obtain a proteinhydrolysatecontaining a mixture of amino acids including leucine. Theprotein hydrolysate, however obtained, is then subjected to theprocedure set forth hereinafter, or its equivalent.

Where hydrochloric acid is used as the hydrolyzing medium, the proteinhydrolysate is concentrated under reduced pressure to remove as much ofthe free hydrochloric acid as possible without raising the temperatureabove 120 C. In this way, about 40% of the original hydrochloric acid isrecovered and may be used in making up, the mix for a subsequenthydrolysis. The thick residue is taken up with about twice its volume ofhot water and partially neutralized by the addition of alkali,preferably a strong solution of sodium hydroxide, although hydrates orcarbonates of any alkali metal or alkaline earth metal may be employed,such as sodium carbonate. This neutralization should be carried to apoint within the hydrogen ion concentration range represented by a pHvalue of 1.8 to 2.6, the particular value chosen depending upon otherfactors such as the amount of tyrosine present in the particular proteinbeing used. The neutralization may be followed and closely controlled bysue of the quinhydrone electrode or by any other method of determininghydrogen ion concentr'a- 40 tion which may be adapted to the purpose. Inmaking these determinations, it may be advisable in some cases topartially decolorize an aliquot portion of the hydrolysate withactivated carbon'to avoid interference by the coloring matter in thesolution. I The mixture is thenpartially decolorized by boiling for afew minutes with activated carbon and filtering while still hot. Thisdecolorizing treatment presents two distinct advantages in the process,in that the carbon is more eflfective as a decolorizing agent when thesolution is partially neutralized and avoids the necessity of performinga dimcult'flltratlon of a strongly acid mixture. The process can, ofcourse, be carried on without the decolorizing step but the product willthen be somewhat discolored or require subsequent treatment.

If the protein material used yields a relatively large amount oftyrosine, another of the amino acids resulting from the hydrolysis ofproteins, as is true of casein, then some of the tyrosine willcrystallize out at this point in the process and may be recovered byfiltration. If, however, the percentage of tyrosine is lower, as in thecase of wheat gluten, then all of it remains in solution. Thehydrolysate is then concentrated under reduced pressure until it issaturated, or nearly so, with sodium chloride formed by theneutralization of hydrochloric acid with the alkali. The solution is nowsaturated with an inorganic salt, namely, sodium chloride and I havefound that leucine may be salted out of such solutions containing it andother amino acids, such as solutions arising from the hydrolysis ofproteins, the leucine being particularly susceptible to such salting outefiect and the other amino acids occurring therewith in proteinhydrolysates or the like tending to remain in solution. In this way, aneffective separation of the leucine from related amino acids may beachieved, such a separation having heretofore been a very difilcult one,as might be expected from the similar properties of the individual aminoacids in protein hydrolysate mixtures. While portions of the other aminoacids will also come down with the leucine substance, these are small inquantity, a precipitate containing as high as 85% of forms of leucinehaving been obtained.

From the aforementioned protein hydrolysate approximately saturated withsodium chloride, leucine commences crystallizing out as dileucinehydrochloride before the above-mentioned concentration is complete, thesalt formed in the neutralization acting as the salting-cut agent.Leucine' has not previously been salted out from protein hydrolysates inthe form of the dileucine hydrochloride, from which leucine itself canreadily be regenerated.

While a useful separation of leucine can be achieved from solutionshaving varying hydrogen ion concentrations, I have found that thesalting-out efiect is greatest at a hydrogen ion concentration of thesolution of about pH 2.4, that it rapidly diminishes as the solution ismade more acid, and that it slowly decreases as the pH value shifts from2.4 to 8.0, or from a strongly acid solution to a slightly basic one. Inthis lastmentioned range, there may be salted out dileucinehydrochloride, mixtures of dileucine hydrochloride and free leucine, orleucine alone, depending upon the hydrogen ion concentration selected.Thus, it is possible to precipitate either form of leucine or mixturesof the two forms in various proportions according to the hydrogen ionconcentration chosen.

However, since the primary obiect of the invention is to secure as largean amount of leucine as can be recovered, as free as possiblefrom-contamination with other amino acids, a pH value as near aspossible to 2.4 should be maintained. The new method is, of course,applicable over a considerable range of hydrogen ion concentration but,in general, satisfactory results may be obtained within a range of pH1.8 to 2.6 and best results at pH 2.4. 'Salting out within this rangegives the best results from the standpoint of yield, purity, utilizationof the mother liquor, etc.

s cm standing 9. Iew hours the dileucine hydroshould be neutralized tochloride is removed by filtration or other suitable means and washedwith 20% sodium chlo ride solution, the resulting product naturallyhaving a high salt content and also containing various amounts of otheramino acids which are carried down in the bulls; precipitate and notentirely removed by washing. This crude dileucine hydrochloride isdissolved in the least volume of hot water possible and neutralized withalkali to about pH 6.0, thus changing the dileucine hydrochloride toleucine, which is less soluble and of which a large portionprecipitates. of crystals is removed and the filtrate concentrated,cooled and .a second crop of crystals recovered.

For a still purer product, the leucine may be dissolved in hot 70%alcohol, decolorized with carbon and allowed to recrystallize. By thispurification step, yields of 2.5% of purified leucine may be obtainedfrom wheat gluten (75-80% protein), yields of 6% from commercial casein,and even more from other proteins.

While the method has been described above with particular reference tothe treatmentof a hydrolysate obtained by treating protein material withhydrochloric acid and then adding sodium hydroxide to neutralize to thedesired pH number and salt out the leucine substance by means of theformed NaCl, leucine and dileucine hydrochloride may be salted out fromprotein hydrolysates with other inorganic salts such as, for example,ammonium sulphate and sodium sulphate, but sodium chloride is preferredbecause it is formed in the hydrolysate as a result of neutralization ofhydrochloric acid used in the hydrolysis. Of course, advantage should betaken of the presence of hydrochloric acid and its ability to formsodium chloride by neutralization with sodium hydroxide, but, where thehydrolysis is not performed with hydrochloric acid, it may sometimes beadvantageous to use other salts for the salting-out step; Also, eitherone salt, alone may be used for this purpose or a mixture of salts maybe used, such a mixture being, for example, the sodium chloride formedin the neutralization of a hydrochloric acid hydrolysate and one of thesulphates mentioned, which may be added in place of additional sodiumchloride for salting out.

The herein described method of obtaining the amino acid, leucine,results in the recovery of a large proportion of this substance presentin the protein, up to within two or three per cent of the amountpresent, and the yields are somewhat proportional to the quantitiesoccurring in the raw material. The leucine thus obtained is quite purebut some latitude in respect to purity may be exercised it a high degreepurity is not desired, as may sometimes be the case. The invention thusincludes the feature of precipitating leucine from protein hydrolysatemixtures con-- taining it, as the also the procedure of maintaining thehydrogen ion concentration of the hydrolysate, or other amino acidmixture, within a preferred range for best results when salting out,although this preferred range may be exceeded while still obtaining aseparation oi. the leucine from the other amino acids presen It isparticularly desirable to maintain the hydrogen ion concentration of thesolution within the'range oi! pH 1.8 to 2.6 where it is expectedsubsequently to treat the same solution further for the isolation ofglutamic acid, for which purpose the solution pH 3.2. Consequently,

This crop.

dileucine hydrochloride, and

addition of. more sodium hydroxide after the separation of leucine isfinished. The glutamic acid is not greatly susceptible to thesalting-out effect and does'not come down in large amount with leucine,its separation being eflected by a diflerent method.

What I claim is: l 1. The method of. treating, a protein selected from agroup consisting of gluten, casein and zein which comprises hydrolyzingthe protein, bringing the hydrolysate to a pH value of from 1.8 to 8.0,in the presence of a sufficient amount of a soluble salt to causeprecipitate.

2. The method of treating a protein selected .i'ro'm a group consistingoi gluten, casein and zein which comprises hydrolyzing the protein withhydrochloric acid, neutralizing the hydrolysate with an alkali to a andconcentrating the mixture to cause the precipitation of a form oileucine.

s. The method of obtaining leucine from its admixture with one or moreof the amino acids leucine.

occurring in the hydrolysate of a protein selected from a groupconsisting of gluten, casein and zein, which comprises adding a solubleinorganic salt to the mixture until the solution is approximatelysaturated with said salt and precipitating out a form of leucine.

4. The method of obtaining leucine from its admixture with one or moreof the amino acids occurring in the hydrolysate of a protein selectedfrom a group consisting of gluten, casein and zein, which compriseshydrolyzing the selected,

protein with hydrochloric acid to form said ad.- mixture of acids, andapproximately saturating the mixture with sodium chloride to salt outdileucine hydrochloride.

5. The method of obtaining leucine from its admixture with one or moreof the amino acids occurring in the hydrolysate oi a protein selectedfrom a group consisting of gluten, casein and zein, which compriseshydrolyzing the selected protein with hydrochloric acid to form saidadmixture of acids, approximately saturating the mixture with asuiiicient amount of soluble salt to salt out the dileucinehydrochloride, collecting the precipitate and redissolving the same,bringing the second solution to approximate neutrality and precipitatingout leucine.

6. The method 01' obtaining leucine from its admixture with one or moreof amino acids occurring in the hydrolysate of a protein selected from agroup consisting oi gluten, casein and zein, which comprises bringingthe mixture to a-pI-I value of about 1.8 to 2.6, and enriching themix-'-v ture in soluble salts to precipitate out a form of '1. Themethod or obtaining leucine from its admixture with one or-more of theamino acidsoccurring in the hydrolysate of a protein selected from agroup consisting of gluten, casein and zein, which comprises bringingthe mixture to a pH vvalue 01' about 1.8 to 2.6, and adding a sumcientquantity oi at least one soluble salt thereto to precipitate out a, formof leucine.

' 8. The method 01 obtaining leucine from: its admixture with one ormore of the amino acid s occurring in the hydrolysate of a proteinselected from a group consisting oi. gluten, casein and a form ofleucine to I admixture with one or more oi the amino acids pH value of1.8 to 8.0,

zein, which comprises neutralizing the mixture to a pH value of about1.8 to 2.6 by the addition or alone, concentrating the mixture andprecipitatingout a form of leucine.

9. The method of obtaining leucine from its admixture with one or more01' the amino acids occurring in the hydrolysate of a protein selectedfrom a group consisting of gluten, casein and zein, which compriseshydrolyzing the selected protein with hydrochloric acid to form saidadmixture oi' acids, neutralizing the mixture to a pH value 01 2.4,concentrating the same, and precipitating out dileucinehydrochloride.

10. The method of obtaining leucine from its occurring in thehydrolysate of a protein selected from a group consisting of gluten,casein and zein, which comprises hydrolyzing the selected protein withhydrochloric acid to form said admixture of acids, neutralizing themixture to a pH value 01' about 2.4, concentrating the same,precipitating out dileucine hydrochloride, dissolving the latter andneutralizing the solution to about pH 6.0 to precipitate leucine.

11. The method oi.'*obtaining leucine from its admixture with one ormore of the amino acids occurring in the hydrolysate of a proteinselected from a group consisting of gluten, casein and zein, whichcomprises hydrolyzing the selected protein with hydrochloric acid toform said ad:- mixture of acids, neutralizing the mixture with sodiumhydroxide to a pH value o 2.4,concentrating the mixture and collectingthe salted out dileucine hydrochloride.

12. The method of obtaining leucine from its admixture with one or moreof the amino acids occurring in the hydrolysate of a protein selectedfrom a group consisting oi gluten, casein and zein, which .comprisesneutralizing the mixtureto a pH value of about 1.8 to 25.6, decolorizingwith activated carbon, filtering, iconcentrating the mixture andcollecting the leucine.

precipitated form of 13. The method 01' obtaining leucine from itsadmixture with one or more of the amino acids occurring in thehydrolysate of a protein selected from a group consisting of gluten,casein and zein, which comprises hydrolyzing theselected protein withhydrochloric acid to form said admlxtureoi. acids, neutralizing themixture with a base until a pH value oi about 1.8 to 2.6 is reached,concentrating the neutralized mixture until approximate saturation withsalt is attained, and collecting the salted out dileucine hydrochloride.

14. The method of obtaining leucine from its admixture with one or moreof the amino'acids occurring in the hydrolysate of a protein selectedfrom agroup consisting of gluten, casein and zein, which compriseshydrolyzing the selected protein with hydrochloric'acid to form saidadmixture 'of acids, bringing the mixture to pH values within the rangefrom 2.4 to 8.0 and concentrating the same in the presence 01' asuincientamotm't of a soluble salt to precipitate out dileucinehydrochloride, mixtures oi dileucine hydrochloride and leucine, orleucine respectively, according as the acidity of the mixture isdecreased.

. HAROLD M. BARNEI'I.

